This project will evaluate the properties of a novel glutathione peroxidase enzyme that is considerably enriched in lungs and plays an important role in lung antioxidant defense and lung surfactant metabolism. This recently described protein called peroxiredoxin 6 (Prdx6 or 1-cys Peroxiredoxin) represents the only non-selenium glutathione peroxidase of relatively high specific activity. An important characteristic in the activity spectrum for this enzyme is its ability, unlike classical glutathione peroxidase, to reduce phospholipid hydroperoxides as, for example, peroxidation of membrane phospholipids during oxidant stress. During the past 4 years of support as a component project of a P-01 grant, we have demonstrated that Prdx6 plays a seminal role in defense of lungs against oxidant stress (hyperoxia, paraquat), that the promoter of the gene contains an antioxidant response element that is sensitive to the transcription factor Nrf2, and have developed a model based on lipid binding studies for the coordination of the peroxidase (Prx) and phospholipase (PLA2) activities of the protein. We are now seeking 5 years of support to extend these studies. Specific Aim 1 will utilize knock-in technology in the Prdx6 null cells to evaluate the respective contributions of the 2 activities (Prx, PLA2) to antioxidant protection. This aim will also directly compare the relative roles of Prdx6 and the other major glutathione peroxidase (GPx1) in antioxidant protection. Specific Aim 2 will study induction of Prdx6 by combined KGF and dexamethasone treatment, which our preliminary data indicate have a synergistic effect on Prdx6 expression. Specific Aim 3 will continue studies to evaluate structure-function characteristics of the protein with a special focus on phospholipid binding as a requirement for the phospholipid hydroperoxide peroxidase activity and the role of Prdx6 post-translational modifications. Specific Aim 4 will utilize cellular systems for analysis of the role of pGST in activation of Prdx6 activity. The proposed studies will provide a coordinated effort to investigate the role of this novel antioxidant enzyme in lung defense against oxidant stress and will provide new information concerning the biochemical regulation of its enzymatic activity. This information could lead to new approaches to increasing the ability of the lung to tolerate oxidant stress.